The Drosophila midline is comprised of a distinct group of cells within the CNS that develop into a diverse set of neurons and glia. These cells play essential roles in CNS development, many of which are analogous to events in vertebrate CNS. The developmental importance of the midline, the large number of genes and pathways involved in midline development, and the diverse morphogenetic events that take place in the midline make this a premiere system for comprehensive and mechanistic developmental studies. Nearly 300 genes have been identified that are expressed in some or all CNS midline cells. The transcription factor Single minded (Sim) is required for specification of the midline and likely regulates most midline genes, but additional regulators are required to explain the diverse temporospatial patterns observed. A small number of midline CRMs have been identified, but mechanisms regulating when and where each gene is expressed are largely unknown. As a necessary step towards comprehensive characterization of midline gene regulation, I will use several approaches to identify important transcriptional mechanisms involved in midline development. I will perform a screen for midline-specific CRMs, by large-scale cloning of genomic regions around midline genes, assayed using Drosophila germline transgenesis of Green Fluorescent Protein and GAL4 reporters. Novel CRMs will be analyzed bioinformatically to identify putative regulatory motifs, and these motifs will be tested in vivo to identify the sequences conferring midline expression. Several previously unknown regulatory mechanisms have already been revealed, and will be studied in detail. I will screen mutants for transcription factors with midline glial roles for regulation of all known glial genes, to provide a global hierarchical view of midline glial regulatory networks. Finally, I will initiate a genome-wide screen for direct Sim using Chromatin Immunoprecipitation/high throughput sequencing. Midline cells will be isolated from embryos at multiple stages, Sim-bound DNA will be isolated by Immunoprecipitation, and DNA will be sequenced to map Sim binding dynamics throughout midline development. This will identify a large set of direct Sim targets that can be analyzed for their functions in the midline, and provide a robust molecular context for studying midline gene regulation. PUBLIC HEALTH RELEVANCE: Detailed analysis of gene regulatory mechanisms underlying nervous system development is crucial to diagnosing and treating diseases that arise when these mechanisms go awry. As many regulatory networks, including those used in the CNS, are shared between invertebrates and vertebrates, insight into these networks will have profound implications in vertebrates. The Drosophila CNS midline is analogous to the vertebrate floorplate, so regulatory and signaling components may be functionally equivalent.